Anti phospho c met

Tumor xenografts were excised and rapidly frozen in OCT compound (Tissue-Tek, Torrance, CA) and tumor tissue sections were cut on a cryostat (Leica, Buffalo Grove, IL). Following standard protocols, the sections were processed for immunolabeling with anti-CD31 (1:50 dilution) (Abcam, Cambridge, MA), anti-phospho-c-Met (1:300 dilution), total-Met (1:300 dilution) (Cell Signaling, Danvers, MA) or anti-HO-1 (1:50 dilution) (Novus Biologicals, Littleton, CO); and subsequently labelled with a species-specific horseradish peroxidase-conjugateed secondary antibody. In tissue sections stained with anti-CD31 antibody, mean vessel density was calculated by standard grid counting method at x400 magnification.

Harvested tumor tissues were quickly frozen using the OCT compound (Tissue-Tek, Torrance, CA). Tissue sections were prepared on a cryostat (Leica, Buffalo Grove, IL). For immunolabeling, the sections were then treated with anti-CD31 (1:50 dilution) (Cat# 550274, BD Pharmingen)), anti-phospho-c-Met (1:300 dilution) (Cat# 3077), anti-total Met (1:300 dilution) (Cat# 8198, Cell Signaling), or anti-4-HNE (1:200 dilution) (Cat# ab46545, Abcam); and subsequently labeled with species-specific horseradish peroxidase-conjugated secondary antibody. Tissue specimens were developed in 3-amino-ethylcarbazole and counterstained with Gills hematoxylin. In anti-CD31-stained sections, the means for vessel densities were calculated by the standard method of grid counting at a magnification of x400^{5 (link),15 (link)}.
The samples were quantitatively analyzed by the software ImageJ using an immunohistochemical composite scoring system or the immunoreactive scoring system (IRS)^{54 (link)}. In this quantification system, the composite score or immunoreactive score provides a range of 0 to 12 as a product of percentage of positive cells score (0–4) and staining intensity score (0–3); and a final score of 9–12 is considered to be strongly positive expression.

c-Met inhibitors (Cabozantinib-#S1119, Crizotinib-S1068, Foretinib-#1111, Capmatinib-#S2788) and CDK4/6 inhibitors (Abemaciclib-#S7158, Palbociclib-#S1116, Ribociclib-#S7440) were purchased from Selleckchem (Houston, TX, USA). The c-Met-targeting antibody, SAIT301 was produced according to the previous literature [22 (link)]. Antibodies for anti-c-Met (#8494S) and anti-phospho-c-Met (#3077S) were purchased from Cell Signaling. Anti-β actin (#ab8227) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Laminin (#L2020) and DMSO (#D2650) were purchased from Sigma Aldrich (St. Louis, MO, USA) and normal human IgG control (#1-001-A) was purchased from R&D Systems (Minneapolis, MN, USA). Hoechest33342 (#H3570) was purchased from Life Technologies (Carlsbad, CA, USA).